A method for in vivo evaluation of α-glucosidase inhibition using Drosophila

The development of α-glucosidase inhibitors is essential for the prevention of type II diabetes. Previous research has investigated in vitro inhibition using isolated α-glucosidase, which may not accurately reflect physical processes. The method presented in this study aims to establish a rapid and inexpensive in vivo method to study the inhibition of α-glucosidase activity using Drosophila as a model organism. This method can be used to calculate the IC50 value of compounds of interest for inhibition of α-glucosidase activity. The method established in this study can be used for in vivo screening of anti-diabetic compounds. • A rapid and inexpensive in vivo method to study the inhibition of α-glucosidase activity.• This method can be used to calculate the IC50 value of compounds of interest for inhibition of α-glucosidase activity.• This is a useful method for in vivo screening of anti-diabetic compounds.


a b s t r a c t
The development of -glucosidase inhibitors is essential for the prevention of type II diabetes.Previous research has investigated in vitro inhibition using isolated -glucosidase, which may not accurately reflect physical processes.The method presented in this study aims to establish a rapid and inexpensive in vivo method to study the inhibition of -glucosidase activity using Drosophila as a model organism.This method can be used to calculate the IC 50 value of compounds of interest for inhibition of -glucosidase activity.The method established in this study can be used for in vivo screening of anti-diabetic compounds.
• A rapid and inexpensive in vivo method to study the inhibition of -glucosidase activity.
• This method can be used to calculate the IC 50 value of compounds of interest for inhibition of -glucosidase activity.• This is a useful method for in vivo screening of anti-diabetic compounds.

Subject area:
Pharmacology, Toxicology and Pharmaceutical Science More specific subject area: Enzyme activity and inhibition assay.Name of your method: Evaluating the inhibition of active compound on -glucosidase activity using Drosophila as a model.

Introduction
Inhibition of -glucosidase is critical for the management and treatment of type 2 diabetes, a disease characterized by elevated blood glucose, insulin resistance, and relative insulin deficiency [3] .By reducing the rate at which carbohydrates are broken down into simple sugars, -glucosidase inhibitors reduce postprandial glucose spikes and help control of blood glucose levels, which is an effective way to treat diabetes [4] .In addition to their role in treating diabetes, -glucosidase inhibitors have also been studied for their potential benefit in the treating other diseases.Some research suggests that slowing carbohydrate digestion may increase feelings of satiety and reduce overall food intake, which in turn would contribute to weight loss [5] .Currently, most studies investigating anti-glucosidase activity are conducted in vitro , conditions that do not fully reflect the complexity of in vivo conditions.Animal studies are needed to identify and validate new, effective pharmacological agents for the treatment and prevention of diabetes.
Drosophila melanogaster is widely recognized as a model organism for the study of metabolic diseases.Drosophila has been used to develop diabetes models that closely resemble the features of type 2 diabetes.A model for the induction of T2D in Drosophila was developed using a high-sugar diet [6] .As in humans, Drosophila contains insulin producing cells (IPCs), insulin-like peptides (dILPs), and an insulin receptor (InR) [7][8] .Insulin-resistant traits such as metabolic abnormalities, elevated Dilp mRNA levels, and impaired insulin signaling activity can be effectively reproduced in both larval and adult stages of Drosophila [9] .Hyperglycemia, insulin resistance, increased fat storage, and shorter lifespan have been studied in Drosophila fed a high-sugar diet [10] .In addition, the effects of plant extracts on preventing metabolic disorders associated with excessive sugar consumption in Drosophila have been studied [11][12][13] .It has been observed that fruit flies contain the enzyme glucosidase for sugar uptake [14] .Therefore, Drosophila serves as a useful model organism for studying the inhibition of -glucosidase activity by compounds of interest.In this study, we developed a protocol for measuring -glucosidase activity using acarbose as a reference compound.This study's methodology offers several advantages over existing approaches.In particular, testing requires only six hours as opposed to the typical seven to ten days, and the IC 50 concentration can be determined, a feature not described in any previously published reports.These benefits make it particularly useful for screening compounds of interest in vivo .In addition, the established protocol provides a rapid and cost-effective in vivo evaluation method for anti-diabetic compounds.

Method details
Preparation of 50 mM potassium phosphate buffer (pH 7.0 at 25 °C): 1. Add 6.15 mL of 1.0 M potassium phosphate dibasic solution to a beaker.2. To the same beaker, add 3.85 mL of 1.0 M potassium phosphate monobasic solution.1.To prepare a high-sugar diet (30% sucrose solution), add 30 g sucrose to a volumetric flask and make up the volume to 100 mL with sterile water.2. To prepare a diet containing 100 M acarbose (MW = 645.604g/mol), add 0.0065 g acarbose to a volumetric flask and then make up the volume to 100 mL with a 30% sucrose solution.For the preparation of diets containing acarbose at concentrations of 5, 2.5, 1.25, 0.625, 0.3125, 0.156, and 0.078 M, the dilution was calculated using the equation

Drosophila strain and culture conditions
The wild-type D. melanogaster strain Oregon-R-C was used in this study.Flies were maintained in a conventional wheat cream medium supplemented with yeast powder and kept in the laboratory at a temperature of 25 ± 1.2 °C and a relative humidity of 70-80% on a 12:12 light/dark cycle.Surviving flies were transferred to fresh food vials every 2 days.

Experimental design and treatment
The experiment was divided into seven groups: 1) high sugar diet group, 2) high sugar diet + 0.078 uM acarbose, 3) high sugar diet + 0.156 uM acarbose, 4) high sugar diet + 0.3125 uM acarbose, 5) high sugar diet + 1.25 uM acarbose, 6) high sugar diet + 2.5 uM acarbose, and 7) high sugar diet + 5 uM acarbose.In each group, the experiment was performed with a total of 100 flies, distributed among 5 vials ( N = 20 per vial).In this study, the negative control group consisted of Drosophila treated with a 6% sugar diet.In this investigation, the concentration of acarbose was determined empirically based on our preliminary analyses.Initial assays utilizing concentrations of 20 uM, 10 uM, and 5 uM revealed inhibition activities in excess of 95%.Following dose reduction to concentrations of 2.5 uM and 1.25 uM, inhibition activities were 81.40 and 68.79%, respectively.As a result, a concentration of 5 uM was regarded optimal for the study's initial acarbose concentration.The details of the protocol are as follows: 5. Separate the flies into vials containing cotton soaked with water to prevent dehydration and starve them for two hours.6.After 2 h of starvation, the flies were transferred to the test and fed for 4 h.7.After the feeding period, the flies were transferred to an empty vial.8.The flies were euthanized with 5% CO 2 fumigation and then placed in the refrigerator at − 20 °C for 10 min.
Preparation of the sample 1.After the flies were euthanized, they were transferred to a 1.5-mL microcentrifuge tube.2. The fly samples were washed three times with 1.0 mL of 50 mM potassium phosphate buffer.3. Fly samples were then homogenized with a pestle in 1.0 mL of 50 mM potassium phosphate buffer using.4. Fly debris was removed from the homogenate by centrifugation at 10,000 rpm for 10 min at 4 °C. 5.The supernatant was transferred to new 1.5 mL microcentrifuge tubes and kept on ice during preparation for measurement of protein content and -glucosidase activity.

Measurement of protein content
1.The Bradford reagent was thoroughly mixed in its container and then brought to room temperature.2. Protein standards were prepared in a buffer containing 0.125 to 1.0 mg/mL BSA.
3. 5 μL of the protein standards or samples were added to separate wells of the 96-well plate.4. 250 μL of Bradford reagent was added to each well and mixed on a shaker for approximately 30 s. 5. Samples were incubated at room temperature for 10 min.The absorbance was then measured at 595 nm. 6.The absorbance of each standard was plotted against its protein concentration.7. The protein concentration of the fly supernatant samples was determined by comparing the net A595 values to the standard curve.

Measurement of 𝛼-Glucosidase activity
1. 50 μL of buffer was added to each well of the 96-well plate.100 μL of buffer was added to the blank wells.2. 50 μL of samples were added to each well of the 96-well plate, including the blank well.
3. 50 μL of 5 mM p -nitrophenyl-d -glucopyranoside was added to each well of the 96-well plate, excluding the blank well.4. The samples were incubated at room temperature for 30 min.Then, the absorbance was measured at 405 nm. 5.The absorbance values of all samples were divided by the protein.
6.The inhibitory activity (I) was calculated according to the following formula: Here, Abs of the control represents the value OD of the sample from the high-sugar diet group.
7. IC 50 values were obtained from plots of percent inhibition versus log concentration of acarbose and calculated from the mean inhibition values using nonlinear regression analysis.

Validation of the method
In this study, we developed a rapid and inexpensive method to investigate the in vivo inhibition of -glucosidase using wildtype female D. melanogaster of the Oregon-R-C strain at 8-10 days of age.To induce -glucosidase activity, Drosophila were treated with a 30% sucrose solution.Acarbose served as a positive control to establish the method.Our results indicate that treatment with the 30% sucrose solution increased -glucosidase activity by 15% compared with the control group treated with a 6% sucrose solution ( N = 100, 20 per vial).The IC 50 value for acarbose-mediated -glucosidase inhibition in Drosophila was determined to be 0.587 ± 0.014 μM ( N = 100, 20 per vial).Thus, this method offers an efficient means of evaluating the potential of anti-diabetic compounds and provides finding that are more relevant to in vivo conditions than in vitro studies ( Figs. 1-4 ).

3 .
Bring the final volume to 200 mL with ultrapure water.4. Adjust the pH to 7.0 at 25 °C with 1 M KOH or HCl.Preparation of the diet solution: